Method for typing of HLA class I alleles

ABSTRACT

This invention provides a method, a kit and a reagent for typing of the HLA class I alleles. Explaining concretely, a single HLA class I antigen or allele is determined by combining PCR amplification using a primer pair which can amplify all HLA-A alleles, all HLA-B alleles or all HLA-C alleles, or which is specific to the common sequence to alleles of the specific group consisting of the specific HLA-A alleles or the specific HLA-B alleles, with reverse hybridization analysis using DNA probes capable of specifically hybridizing with the sequence of al least a specific HLA-A allele, at least a specific HLA-B allele or at least a specific HLA-C allele, which are covalently immobilized on wells of microtiter plates.

TECHNICAL FIELD

HLA (Human Leukocyte Antigen) that is Human major histocompatibilityantigen, is expressed on membranes of imuunocompetent cells, presentsprocessed peptides derived from exogenous and endogenous antigens to Tlymphocytes, and functions as a marker to recognize self and non-self.The present invention relates to a method, a reagent and a kit fortyping of the HLA class I alleles. This invention is especially usefulfor judgement of compatibility between a donor and a recipient in organtransplantation, and for association analysis between the HLA class Igenes and various types of diseases in the clinical and medical field.This invention enables us to easily automate and mechanize detection anddetermination of the HLA class I alleles.

BACKGROUND OF ART

Typing of the HLA antigens has been mainly performed by the serologicalmethod using human alloantibodies. By using the specific antibodies toeach HLA antigen which are contained in cord blood or serum fromsubjects who have frequently undergone blood transfusion,complement-mediated cytotoxicity is caused in the antigen-antibodyreaction. It changes permeability of positive cell membranes to take aneosinic pigment into the cell, resulting in being detected as coloredand expanding cells with a microscope. It is possible to type HLA-A,HLA-B and HLA-C antigens belonging to HLA class I, and HLA-DR and HLA-DQantigens belonging to HLA class II by this method. However, this methodhas problems in terms of collection, quality control and supply of thespecific antibodies. Furthermore, the survival rate of cells is utilizedas an indicator for judgement in this method. Therefore, poor conditionsof subjects, for example, a low survival rate of cells caused by diseaseor influence by passage of time after blood collection, lead to decreaseof credibility for results of testing.

In recent years, a development of molecular biotechnology has enabled usto analyze the region of genes encoding the HLA antigens. That hasclarified the correspondence between the HLA antigens and the sequencesof the HLA genes. This means it has been possible to identify the HLAantigen type by analyzing the specific sequences of the HLA genes (DNAtyping). Especially, PCR (polymerase chain reaction) method which canhigh-sensitively detect a slight change of sequences is utilized to typethe HLA-DR, -DQ, or -DP genes belonging to HLA class II. SeveralPCR-based typing methods for HLA class II DNA such as PCR-SSOP(Sequence-Specific Oligonucleotide Probe) method, PCR-RFLP (RestrictionFragment Length Polymorphism) method, PCR-SSP (Sequence-SpecificPrimers) method and PCR-SSCP (Single Strand Conformation Polymorphism)method have been developed. In all these methods, the gene region toanalyze is amplified by the PCR method and then the variable region inthe sequences of the amplified products is analyzed by combination withanother methods in order to distinguish the genotype. The HLA class IIDNA typing method makes it possible to classify the HLA type at theallele level in addition to classification by the classical serologicalmethod using human alloantisera.

Development of the PCR based-method for HLA class I DNA typing isdelayed remarkably, comparing with HLA class II typing. The reasons areas follows: (1) While almost all the class II gene mutations (genesubstitutions), including those which reflect the specificity ofantigens, concentrate in the region of the exon 2, the class I genemutations are interspersed among the regions of the exons 2 and 3, orthe exon 4. (2) The HLA class I genes, including non-classical genes(HLA-E, -F and -G) and pseudogenes (HLA-H, -J, -K and -L), are highlyhomologous among them.

To date, several HLA class I DNA typing methods have been reported.However, all these methods require complicated manipulation, strictreaction condition and skill. Those are not suitable for handling alarge number of samples and offer only low resolution HLA typing.Furthermore, the typing methods for each gene are not standardized.

DISCLOSURE OF INVENTION

The purpose of this invention is to solve problems of the manipulationof HLA class I locus antigen typing by the classical serological method,and to prodive a method, a kit and a reagent for classifing the subtypeof the HLA class I antigens at the allele level (allele typing), whichhas not been distinguished by the classical method. Furthermore, the aimof this invention is to provide a method for typing of the HLA class Ialleles which can automate and machanize easily.

As a result of intensive studies for these subjects, the inventors haveestablished primers which can amplify all the HLA-A alleles, all theHLA-B alleles or all the HLA-C alleles and specific primers to thecommon sequences among all alleles in the group consisting of thespecific HLA-A alleles or the specific HLA-B alleles. The inventors haveestablished probes which can specifically hybridize with the sequence ofat least one specific HLA-A allele, at least one specific HLA-B alleleor at least one specific HLA-C allele. The inventors have found out thatit is possible to distinguish the HLA class I antigen or allele, byhybridizing the PCR amplified products derived from the specific HLAclass I allele or the specific group with the DNA probes described abovewhich are immobilized on wells of microtiter plates, adding anenzyme-conjugate which can specifically bond to a label of the amplifiedproducts at the same time as or after the hybridization, and adding achromogenic substrate, a luminescent substrate or a fluorescentsubstrate to the mixture, to detect as signals whether or not theamplified products are hybridized with the immobilized DNA probes. Thus,they have accomplished this invention.

The main embodiment of this invention is a method for typing of HLAclass I alleles, which comprises the following steps from (a) to (d).

-   -   (a) A step, using HLA class I gene or nucleic acids containing        their fragment for a template,        -   (1) To non-selectively amplify all HLA-A alleles, all HLA-B            alleles or all HLA-C alleles by a PCR method using a primer            pair which can amplify all the HLA-A alleles, all the HLA-B            alleles or all the HLA-C alleles, or        -   (2) To selectively amplify a specific group consisting of            specific HLA-A alleles or specific HLA-B alleles by a PCR            method using a primer pair which is specific to the common            sequence to alleles of the specific group consisting of the            specific HLA-A alleles or the specific HLA-B alleles,    -   (b) A step to add the above products amplified by the PCR method        to wells of microtiter plates, wherein each well is modified        with a carboxyl group to covalently immobilize amino-modified        DNA probes which can specifically hybridize with the sequence of        at least one specific HLA-A allele, at least one specific HLA-B        allele or at least one specific HLA-C allele, and to hybridize        the amplified products with the immobilized DNA probes, wherein        the DNA probes are selected depending on the above amplified        specific HLA class I gene or group;    -   (c) A step to detect as signals whether or not the amplified        products are hybridized with the immobilized probes; and    -   (d) A step to determine the type of the HLA class I allele based        on the signal pattern detected at the step (c) according to the        Typing Table.

The PCR amplification of the target gene at the step (a), can beclassified into 2 steps. One is a step to non-selectively amplify allthe HLA-A alleles, all the HLA-B alleles or all the HLA-C alleles by thePCR method using a primer pair which can amplify all the HLA-A alleles,all the HLA-B alleles or all the HLA-C alleles. The other is a step toselectively amplify the specific group consisting of the specific HLA-Aallele group or the specific HLA-B allele group by the PCR method usinga primer pair which is specific to the common sequences to alleles ofthe specific group consisting of the specific HLA-A alleles or thespecific HLA-B alleles. At the former step, PCR primers are designed tobe specific to the common sequences, which are within the region of allalleles belonging to the HLA-A allele, the HLA-B allele or the HLA-Callele, or ahead and behind the region. At the latter step, PCR primersare designed to be specific to the common sequences to all allelesincluded in the specific group in order to amplify the specific group.When the specific group is selectively amplified in the presence of somegroups, the primers described above don't need to be used for both asense primer and an antisense primer of a primer pair corresponding tothe specific group. It is possible to use the specific primer to thespecific group for one of primers and the specific primer to all thegroups for the other. The latter step can be performed according to thereference described by the inventors (Tissue Antigens 1997, Vol.50,535-545). A method to selectively amplify alleles encoding the HLA-A2antigen or the HLA-B40 antigen as a group is disclosed in the presentdescription.

At the step (a), the PCR-amplified products derived from the allelebelonging to the HLA-A alleles, the HLA-B alleles or the HLA-C alleles,or from the specific group, are produced. But it is not possible todistinguish the type of the HLA class I allele at the step. Thehybridization reaction at the step (b) using the specific DNA probes isapplied to the following steps.

The Typing Table at the step (d) is made using signal patterns obtainedby hybridizing the PCR amplified products from samples whose HLA class Iantigen types or allele types are known, with DNA probes which canspecifically hybridize with the sequence of at least one specific HLAclass I allele. Persons skilled in the art can make easily the TypingTable. As the Typing Table, FIGS. 1 to 6 can be referred. If someonewants to use DNA probes, which are not described in this description,another Typing Table can be used. The Typing Table is made from signalpatterns obtained by hybridizing the PCR amplified products from sampleswhose HLA class I antigen types or allele types are known, with anotherDNA probe. As described above, persons skilled in the art can also makeeasily these Typing Tables. It should be considered that each sample hasthe HLA class I allele type in a homozygous or heterozygous state, whenthe HLA class I allele type is distinguished according to the TypingTables.

In a perferable embodiment, the PCR method at the step (a) is performedby using a primer pair in which at least one of them is labeled, inorder to detect whether or not the amplified products hybridize withimmobilized DNA probes as signals at the step (c) described above. Inthe other embodiment, the above PCR can be performed by using 4 kinds ofdeoxyribonucleotide triphosphate (dNTP) in which at least one of them islabeled. As a substance used for labeling, a radioisotopic substance, ora non-radioisotopic substance such as a biotin or a digoxigenin, can beutilized.

In a preferable embodiment, at the step (b) or (c) described above, thehybridization of the products amplified by the PCR method with theimmobilized DNA probe is performed by addiing an enzyme-conjugate whichcan specifically bond to a label of the amplified products is added atthe same time as hybridization or after, and the amplified productshybridizing with the immobilized DNA probe is detected as signals byadding a chromogenic substrate, a luminescent substrate or a fluorescentsubstrate which can specifically react with the enzyme. When aperoxidase-conjugated streptavidin is used as an enzyme-conjugate, thesignal can be immediately detected after washing by adding anenzyme-conjugate at the same time as hybridization.

In a preferable embodiment, at least one of a primer pair at the step(a) described above is biotinylated, and an enzyme-conjugate which canspecifically bond to the biotinylated label at the step (b) or (c) is anenzyme-conjugated streptavidin, for example, a peroxydase-conjugatedstreptavidin or an alkaline phosphatase-conjugated streptavidin.

In a preferable embodiment, the hybridization of the products amplifiedby the PCR method with immobilized DNA probes is performed in a solutioncontaining formamide at the step (b) described above. The formamideconcentration of the solution described above (hybridization buffer) isfrom 5% to 30%, and from 10% to 25% as a preferable concentration. Theconcentration can be changed according to the sequence, the length andthe type of the used DNA probe. The most preferable formamideconcentration is about 20%.

In a preferrable embodiment, the hybridization at the step (b) isperformed in a solution containing formamide at the temperature of the37° C. The preferable temperature is from 32° C. to 42° C. Thetemperature can be changed according to the sequence, the length and thetype of the used DNA probe as mentioned above for the formamideconcentration. The most desirable temperature is about 37° C.Hybridization is usually performed at comparatively high temperature, atabout 65° C., to improve the specificity. By using the solutioncontaining formamide, the reaction can be performed at low temperature,at about 37° C.

In a preferable embodiment, when the solution containing formamide isused for the hybridization at the step (b) described above, thetemperature for washing after hybridization of the amplified products bythe PCR method with immobilized DNA probes and/or after binding a labelof the amplified products with an enzyme-conjugate is performed at roomtemperature. Namely, washing can be performed at low temperaure likeroom temperature as by using the solution containing formamide, as wellas the above hybridization.

The amino-modified DNA which can specifically hybridize with at leastone specific HLA-A allele, used at the step (b) in this invention, canbe selected from the group consisting of A98T (SEQ ID No.:1), A98A (SEQID No.:2), A160A (SEQ ID No.:3), A239A (SEQ ID No.:4), A238A (SEQ IDNo.:5), A240T (SEQ ID No.:6), A257TC (SEQ ID No.:7), A259AC (SEQ IDNo.:8), A270T (SEQ ID No.:9), A282C (SEQ ID No.:10), A290T (SEQ IDNo.:11), A299T (SEQ ID No.:12), A302G (SEQ ID No.:13), A355G (SEQ IDNo.:14), A362TA (SEQ ID No.:15), A362TT (SEQ ID No.:16), A368A (SEQ IDNo.:17), A368G (SEQ ID No.:18), A368T (SEQ ID No.:19), A402G (SEQ IDNo.:20), A423T (SEQ ID No.:21), A448C (SEQ ID No.:22), A485A (SEQ IDNo.:23), A524G (SEQ ID No.:24), A526T (SEQ ID No.:25), A527A (SEQ IDNo.:26), A538CG (SEQ ID No.:27), A539A (SEQ ID No.:28), A539T (SEQ IDNo.:29), A555T (SEQ ID No.:30), A559G (SEQ ID No.:31), A570CG (SEQ IDNo.:32), A570GT (SEQ ID No.:33), A779A (SEQ ID No.:34), A843A (SEQ IDNo.:35), A34 (SEQ ID No.:100), A282CT (SEQ ID No.:101), A290TR (SEQ IDNo.:102), A302GR (SEQ ID No.:103), A414A (SEQ ID No.:104), A468T (SEQ IDNo.:105), A489A (SEQ ID No.:106), A502C (SEQ ID No.:107), A538TG (SEQ IDNo.:108) and complementary strands thereof.

The amino-modified DNA probe which can specifically hybridize with atleast one specific HLA-B allele can be selected from the groupconsisting of BL1 (SEQ ID No.:36), BL3 (SEQ ID No.:37), BL4 (SEQ IDNo.:38), BL5 (SEQ ID No.:39), BL9 (SEQ ID No.:40), BL10 (SEQ ID No.:41),BL11 (SEQ ID No.:42), BL24 (SEQ ID No.:43), BL25 (SEQ ID No.:44), BL34(SEQ ID No.:45), BL35 (SEQ ID No.:46), BL36 (SEQ ID No.:47), BL37 (SEQID No.:48), BL38 (SEQ ID No.:49), BL39 (SEQ ID No.:50), BL40 (SEQ IDNo.:51), BL41 (SEQ ID No.:52), BL42 (SEQ ID No.:53), BL56 (SEQ IDNo.:54), BL57 (SEQ ID No.:55), BL78 (SEQ ID No.:56), BL79 (SEQ IDNo.:57), BL222A (SEQ ID No.:58), BL272GA (SEQ ID No.:59), BL226G (SEQ IDNo.:60), BL292G (SEQ ID No.:61), BL292T (SEQ ID No.:62), BL361G (SEQ IDNo.:63), BL409T (SEQ ID No.:64), BL512T (SEQ ID No.:65), BL538CG (SEQ IDNo.:66), BL538G (SEQ ID No.:67), BL39R (SEQ ID No.:109), BL50 (SEQ IDNo.:110), BL77 (SEQ ID No.:111), BL272A (SEQ ID No.:112), BL263T (SEQ IDNo.:113), BL527A (SEQ ID No.:114), BL570GT (SEQ ID No.:115) andcomplementary strands thereof.

The amino-modified DNA probe which can specifically hybridize with atleast one specific HLA-C allele can be selected from the groupconsisting of CC (SEQ ID No.:68), A-12 (SEQ ID No.:69), A-2 (SEQ IDNo.:70), A-3 (SEQ ID No.:71), A-4 (SEQ ID No.:72), A-54 (SEQ ID No.:73),B-1 (SEQ ID No.:74), B-2 (SEQ ID No.:75), C-12 (SEQ ID No.:76), C-24(SEQ ID No.:77), C-33 (SEQ ID No.:78), C-43 (SEQ ID No.:79), 134-g (SEQID No.:80), 134-A2 (SEQ ID No.:81), 353TCA1 (SEQ ID No.:82), 343A (SEQID No.:83), RA-2 (SEQ ID No.:116), RA-41 (SEQ ID No.:117), RB-28 (SEQ IDNo.:118), 201gl (SEQ ID No.:119), C206gR (SEQ ID No.:120), R341A (SEQ IDNo.:121), R343g3 (SEQ ID No.:122), 353TCC (SEQ ID No.:123), 361T1 (SEQID No.:124), 361T368g (SEQ ID No.:125), 361T368T1 (SEQ ID No.:126), 369C(SEQ ID No.:127), 387g1 (SEQ ID No.:128), 526AC2 (SEQ ID No.:129),538gAC (SEQ ID No.:130) and complementary strands thereof.

This invention also comprises the DNA probe itself (from SEQ ID No.: 1to SEQ ID No.:83 and from SEQ ID No.:100 to SEQ ID No.:130) which canspecifically hybridize with at least one specific HLA-A allele, at leastone specific HLA-B allele or at least one specific HLA-C allele forusing the method for distinguishing the HLA class I allele type.

Both an amino-modified DNA probe and an unmodified DNA probe can beused. However, when the probe is covalently immobilized on wells ofcarboxylate-modified microtiter plates, the amino-modified probe must beused. Some bases can be deleted from or added to the end of the DNAprobe within the range that the DNA probe can specifically hybridizewith at least one specific HLA-A allele, at least one specific HLA-Ballele or at least one specific HLA-C allele, namely, within the rangethat the DNA probe can keep the original specificity of hybridization.Accordingly, the DNA probes in this invention also comprise DNA probeswtherein some bases are deleted from or added to the nucleic acidsequence from SEQ ID No.:1 to SEQ ID No.:83 and SEQ ID No.:100 to SEQ IDNo.:130 within the range described above.

The primers which can amplify all the HLA-A alleles, all the HLA-Balleles or all the HLA-C alleles at the step (a) in this invention, canbe selected from the group consisting of CGA011 (SEQ ID No.:90), CGA012(SEQ ID No.:91), AIn3-66C (SEQ ID No.:92), 5BCIn37-34C (SEQ ID No.:96),5BCIn37-24g (SEQ ID No.:97) and 5BCIn37-34g2 (SEQ ID No.:99). The primerwhich is specific to the common sequence to alleles of the specificgroup consisting of the specific HLA-A alleles or the specific HLA-Balleles, can be selected from A2-5T (SEQ ID No.:84), A3-273T (SEQ IDNo.:85), A4-8C (SEQ ID No.:86), A4-254G (SEQ ID No.:87), BASF-1 (SEQ IDNo.:88), and BASR-1 (SEQ ID No.:89). This invention comprises the primeritself described above (from SEQ ID No.:88 to SEQ ID No.:92, from SEQ IDNo.:96 to SEQ ID No.:97 and SEQ ID No.:99), used for the method to typethe HLA class I alleles.

Novel HLA-A alleles, HLA-B alleles and HLA-C alleles have beendiscovered. In the report of the WHO (World Health Organization)Nomenclature Committee for the HLA system, 82, 186, and 42 of alleleshave been assigned for the HLA-A, -B and -C loci, respectively, at March1997. This invention can discriminate all these alleles. Furthermore,the method shown in this invention, together with an optional,easy-performed improvement, such as adding extra DNA probes or primers,can cope with discrimination of alleles which may be discovered andenrolled in the future.

This invention can provide a kit and a reagent for typing of the HLAclass I alleles described in this description. Furthermore, thisinvention can provide a kit and a reagent which comprise the DNA probesand the primers described in this description. For example, the kit cancomprises a solution containing the primers (from SEQ ID No.:84 to SEQID No.:92, from SEQ ID No.:96 to SEQ ID No.:97 and SEQ ID No.:99) whichis disclosed in this invention, PCR buffer solution, which may beconcentrated solution, dNTPs, thermostable DNA polymerase, the DNAprobes (from SEQ ID No.:84 to SEQ ID No.:92, from SEQ ID No.:96 to SEQID No.:97 and SEQ ID No.:99) which is disclosed in this invention or amicrotiter plate on whose wells the DNA probes are covalentlyimmobilized, a denature solution, a hybridization buffer, a washingsolution and an instruction for the kit which includes the TypingTables. The primer described above can optionally be labeled with aradioisotopic or non-radioisotopic substance. The primers can form aprimer pair. The solution containing the primer can be freeze-dried.When the primer is not labeled, at least one of four kinds of dNTPs mustbe labeled. When a non-radioisotopic substance is used as a label, anenzyme-conjugate solution, a chromogenic reagent including a chromogenicsubstrate and a chromogenic solution, a luminescent reagent or afluorescent reagent, a stop solution and so on can be added as acomponent in the kit. Furthermore, a component such as guanidinethiocyanate buffer for isolation of genome DNAs, can be optionally addedin the kit to the degree promoting enforcement of this invention.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 indicates a Typing Table showing the reaction pattern betweensamples which the HLA-A2 allele type is known and DNA probes in thepresent invention. Each name of DNA probes is shown on the top in theFigure, and each type of the HLA-A2 alleles is shown on the left side inthe Figure. Closed square and Open square mean a positive and a negativereaction, respectively.

FIG. 2 indicates a Typing Table showing the reaction pattern betweensamples which the HLA-B40 allele type is known and DNA probes in thepresent invention. Each name of DNA probes is shown on the top in theFigure, and each type of the HLA-B40 alleles is shown on the left sidein the Figure. Closed square and Open square mean a positive and anegative reaction, respectively.

FIG. 3 indicates a Typing Table showing the reaction pattern betweensamples which the HLA-A antigen and allele type are known, and DNAprobes in the present invention. Each name of DNA probes is shown on thetop in the Figure, and each type of the HLA-A antigens and alleles isshown on the left side in the Figure. Closed square and Open square meana positive and a negative reaction, respectively.

FIGS. 4 and 5 indicate Typing Tables showing the reaction patternbetween samples which the HLA-B antigen and allele type is known, andDNA probes in the present invention. Each name of DNA probes is shown onthe top in the Figures, and each type of the HLA-B antigens and allelesis shown on the left side in the Figures. Closed square and Open squaremean a positive and a negative reaction, respectively.

FIG. 6 indicates a Typing Table showing the reaction pattern betweensamples which the HLA-C antigen and/or allele type is known, and DNAprobes in the present invention. Each name of DNA probes is shown on thetop in Figure, and each type of the HLA-C and/or alleles is shown on theleft side in Figure. Closed square and Open square mean a positive and anegative reaction, respectively.

THE BEST MODE FOR CARRYING OUT THE INVENTION

The strategy of this invention described above is explained in moredetail.

The typing method in this invention can be explained, dividing into thefollowing 6 steps.

-   -   1) Extraction of chromosome(genome) DNAs,    -   2) PCR amplification of target genes,    -   3) Immobilization of DNA probes on wells of microtiter plates,    -   4) Hybridization of PCR products with DNA probes,    -   5) Detection of signals, and    -   6) Determination of the allele type.        1) Extraction of Chromosome (Genome) DNAs

A method for preparation of genome DNAs is explained as follows.Leukocytes are isolated from collected blood according to usual methodsand are lysed in a guanidine thiocyanate buffer. Proteins are eliminatedby phenol extraction. A sodium acetate buffer (pH 5.2) is added andmixed. Genome DNAs are obtained by adding chilled ethanol.

2) PCR Amplification of Target Genes

The region containing the HLA class I allele is amplified by the PCRmethod using genome DNAs described above for a template. Commercializedreagents can be used for amplification described above. Amplificationcan be performed according to attached instructions. If it is necessary,reaction temperature, reaction time, the number of cycles and so on canbe changed. Then, the amplification is performed by using a primer pairfor a reaction tube. Amplification by adding multiple primer pairs intothe same reaction tubes, can decrease operation task or cost. From theviewpoint of the purpose of this invention, a primer pair which one ofthem is biotinylated, is used for the practical testing or a kit.

For example, A2-5T and 5′-biotinylated A3-273T can be used for a primerpair to amplify the region containing the exon 2, the intron 2 and theexon 3 of the HLA-A2 alleles by the PCR method. A4-8C and5′-biotinylated A4-254G can be used for a primer pair to amplify theregion containing the exon 4 of the HLA-A alleles by the PCR method.These primers are described in the reference of the inventors (TissueAntigens 1997 described above).

For example, BASF-1 and 5′-bitinylated BASR-1 can be used for a primerpair to amplify the region containing the exon 2, the intron 2 and theexon 3 of the HLA-B40 alleles by the PCR method.

For example, CGA011 or CGA012, and 5′-biotinylated AIn3-66C can be usedfor a primer pair to amplify the region containing the exon 2, theintron 2 and the exon 3 of all the HLA-A alleles by the PCR method.

For example, 5BIN1-TA (SEQ ID No.:93) or 5BIN1-CG (SEQ ID No.:94) and5′-biotinylated 3BIN3-37 (SEQ ID No.:95) can be used for a primer pairto amplify the region containing the exon 2, the intron 2 and the exon 3of all the HLA-B alleles by the PCR method. The primers are described inthe reference of Cereb N. et al (Tissue Antigens 1997, Vol.50, 74-76).

For example, 5BCIn37-34C, 5BCIn37-24g or 5BCIn37-34g2, and5′-biotinylated 3BCIn3-12 (SEQ ID No.:98) can be used for a primer pairto amplify the region containing the exon 2, the intron 2 and the exon 3of all the HLA-C alleles by the PCR method. The primer, 3BCIn3-12, isdescribed in the reference of Cereb N. et al (Tissue Antigens 1995,Vol.45, 1-11)

3) Immobilization of DNA Probes on Wells of Microtiter Plates

Amino-modified DNA probes (1-20 pmol) which can specifically hybridizewith the sequence of at least one specific HLA-A allele, at least onespecific HLA-B allele or at least one specific HLA-C allele, are addedonto each well of carboxylate-modified polystyrene microtiter plates andimmobilized covalently by inducing the chemical amino-binding reactionusing a suitable catalyst, for example,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Afterimmobilization of the DNA probes on wells, microtiter plates are washedwith a suitable buffer. After washing, microtiter plates can be storedover an extended period of time on wet and cold condition.

4) Hybridization of PCR Products with DNA Probes

The PCR amplified products are denatured to a single strand DNA understrong alkali, for example, NaOH, and are hybridized with DNA probeswhich are immobilized on wells of microtiter plates. The hybridizationis performed in a solution containing about 20% formamide onhybridization condition at about 37° C. After the hybridization,excessive amplified products or those which don't have the specificsequence to DNA probes described above, are eliminated. DNA probes usedat this step are selected in compliance with the specific HLA class Igene or the specific group which are amplified at the above step.

For example, as for the amplified products from the region containingthe exon 2, the intron 2 and the exon 3 of the HLA-A2 alleles by aprimer pair described above, A2-5T and A3-273T, or the amplifiedproducts from the exon 4 of the HLA-A alleles by a primer pair, A4-8Cand A4-254G, the hybridization can be performed by using A98T, A98A,A160A, A240T, A270T, A290T, A355G, A362TA, A362TT, A368A, A368G, A368T,A402G, A485A, A527A, A539A, A539T, A559G, A570CG, A779A or A843A for DNAprobes.

For example, as for the amplified products from the region containingthe exon 2, the intron 2 and the exon 3 of the HLA-B40 alleles by aprimer pair described above, BASF-1 and BASR-1, the hybridization can beperformed by using BL4, BL5, BL24, BL25, BL34, BL35, BL37, BL39, BL41,BL50, BL56, BL57, BL222A, BL409T or BL512 for DNA probes.

For example, as for the amplified products from the region containingthe exon 2, the intron 2 and the exon 3 of all the HLA-A alleles by aprimer pair described above, CGA011, CGA012 or AIn3-66C, thehybridization can be performed by using A34, A239A, A238A, A257TC,A259AC, A282C, A282CT, A290TR, A299T, A355G, A414A, A448C, A468T, A489A,A502C, A526T, A538CG, A538TG, A539A, A539T, A555T, A570CG, A570GT orA302GR for DNA probes.

For example, as for the amplified products from the region containingthe exon 2, the intron 2 and the exon 3 of all the HLA-B alleles by aprimer pair described above, 5BIN1-TA, 5BIN1-CG or 3BIN3-37, thehybridization can be performed by using BL1, BL3, BL4, BL9, BL10, BL11,BL34, BL36, BL37, BL38, BL39R, BL40, BL41, BL42, BL77, BL78, BL79,BL226G, BL263T, BL272A, BL527A, BL538CG, BL538G or BL570GT for DNAprobes.

For example, as for the amplified products from the region containingthe exon 2, the intron 2 and the exon 3 of all the HLA-C alleles by aprimer pair described above, 5BCIn37-34C, 5BCIn-37-24g, 5BCIn37-34g2 or5BCIn3-12, the hybridization can be performed by using 201g1, C206gR,A-12, RA-2, A-3, RA-41, A-54, B-1, RB-28, C-12, C-24, C-33, C-43, 134-g,134-A2, 353TCA1, 343A, R341A, R343g3, 353TCC, 361T1, 361T368g,361T368T1, 369C, 387g1, 526AC2 or 538gAC for DNA probes.

About the concrete type of the HLA class I allele which aredistinguished by the hybridization with these DNA probes, examples andFigures can be referred.

Besides these DNA probes, A302G, A423T, A524G, BL272GA, BL292G, BL292T,BL361G, CC, A-2, A-4 or B-2 can be used for typing of the HLA class Iantigens or alleles described below. A302G, A423T and A524G canspecifically hybridize with the sequence of the HLA-A antigens oralleles, A*2501 and A*3201, A*2501, A26, A34, A*4301 and A66, andA*2301, A29, A*31012, A*3201, A33 and A*7401,respectively. BL272GA,BL292G, BL292T and BL361G can specifically hybridize with the sequenceof the HLA-B antigens or alleles, B14, B38 and B39, B7, B8, B14, B27,B39, B*4201, B*4601, B*5401, B55, B56, B67, B*7301, B*7801 and B*8101,B13, B15, B18, B35, B37, B38, B40, B41, B44, B*4501, B*4701, B48,B*4901, B*5001, B51, B52, B*5301, B57, B58, B*5901 and B*7802, and B57,respectively. CC can hybridize with the sequence of all the HLA-Calleles. A-2, A-4 and B-2 can specifically hybridize with the sequenceof the HLA-C antigens or alleles, Cw2, Cw3, Cw*0403 and Cw15, Cw*0602,Cw7 and Cw18, and Cw1, Cw3, Cw7, Cw8, Cw*1202, Cw*1203, Cw*1301, Cw*14,Cw*1601 and Cw*16041, respectively.

5) Detection of Signals

An example for detection of signals is explained below. The PCRamplified products hybridizing with DNA probes can be detected byutilizing a label, which they have in themselves, such as a biotin.After an alkaline phosphate-conjugated streptavidin or aperoxidase-conjugated streptavidin which can bond to a biotin, is addedto each well of the microtiter plates, and the plates are sealed, thereaction is performed by incubation on proper temperature condition. Thehybridizing amplified products are detected as signals by using achromogenic substrate such as p-nitrophenylphosphate (PNPP) or3,3′,5,5′-tetramethylbenzidine (TMB). Detection of signals is performedby measurement of the absorbance. The signals described above can beautomatically detected by using a machine, and those by colordevelopment can be easily detected by the naked eye.

6) Determination of the Allele Types

By signal patterns which are detected on the microtiter plate describedabove, for example, in compliance with the Typing Tables which aredisclosed in FIGS. 1-6, the HLA lass I alleles are determined. Patternsof these Typing Tables in FIGS. 1-6 can be arranged in case ofnecessity.

EXAMPLES

This invention is explained in more detail by showing examples, whichare actually performed by using samples, whose HLA types are known.However, the range of this invention is not limited to only theseexamples.

Example 1 HLA-A2 Allele Typing

Leukocytes (Samples 1-4) which were isolated from peripheral blood(about 10 ml) of normal subjects according to usual methods, were lysedin 500 μl of guanidine thiocyanate buffer (4M guanidine thiocyanate, 25mM sodium citrate(pH7.0), 0.5% sodium N-lauroylsarcosinate, 1%mercaptoethanol). The solution was extracted twice with phenol toeliminate proteins. After mixing with 3M sodium acetate buffer (pH 5.2),genome DNAs were obtained by adding twice volume of chilled ethanol. Byusing this DNAs, typing of the HLA-A2 alleles was performed as follows.

By using A2-5T and 5′-biotinylated A3-273T for a primer pair,amplification of the region containing the exon 2, the intron 2 and theexon 3 of the HLA-A2 alleles from DNAs described above was performed bythe PCR method. Likewise, by using A4-8C and 5′-biotinylated A4-254G fora primer pair, amplification of the region containing the exon 4 of theHLA-A alleles was also performed by the PCR method. The reactionsolution was composed of genomic DNAs (100 ng), 1.4 units ofthermostable DNA polymerase which was pretreated with Taq Start™Antibodyfor 5 min at room temperature, 67 mM Tris-HCl (pH 8.8), 16.6 mM ammoniumsulfate, 1.5 mM magnesium chloride, 0.01% Tween 20, 200 μM dNTPs, andeach 1.7 μM of a primer pair in a final volume of 80 μl. DNAamplification was performed by using GeneAmp PCR system 9600 (PerkinElmer) by initial denaturation at 95° C. for 2 min followed by 5 cyclesof denaturation for 25 s, annealing at 70° C. for 45 s, extension at 72°C. for 45 s followed by 36 cycles of denaturation for 25 s, annealing at65° C. for 50 s, extension at 72° C. for 45 s.

5′-amino-modified DNA probes, A98T, A98A, A160A, A240T, A270T, A290T,A355G, A362TA, A362TT, A368A, A368G, A368T, A402G, A485A, A527A, A539A,A539T, A559G, A570CG, A779A and A843A, were immobilized covalently onwells of carboxylate-modified polystyrene microtiter plates as follows.Twenty-five μl of the DNA probes described above which were dissolvedwith sterile distilled water, was added to each of 20 wells which wereused for a sample, in order shown in FIG. 1. Next, 75 μl of 0.2M1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was added to eachwell and mixed. After the plates were sealed and incubated for 16 hoursat room temperature, they were washed four times with PBS buffer (7.5 mMdi-potassium hydrogenphosphate, 2.5 mM potassium dihydrogenphosphate,0.15M sodium chloride). Two hundreds μl of 0.4N NaOH were added to eachwell and the plates were incubated for 1 hour at 37° C. The plates werewashed four times with PBS buffer.

One hundred μl of GMC buffer for hybridization (0.25M di-sodiumhydrogenphosphate, 7% SDS, 1% BSA, 0.5M EDTA, 0.03M phosphoric acid, 20%formamide) was added to each well of the microtiter plates and theplates were incubated for 5 min at 37° C. After incubation, the bufferwas removed from each well. During incubation, 72 μl of the amplifiedproducts which were obtained from the region containing the exon 2, theintron 2 and the exon 3, and 8 μl of the amplified products which wereobtained from the region containing the exon 4, were denatured with anequivalent volume of 0.4 NaOH for 5 min at room temperature. Afterdenaturation, 1800 μl and 200 μl of hybridization buffer were added tothe denatured products, respectively, mixed and 100 μl of them was addedto each well (the former was added Well 1 to Well 18. The latter wasadded to Well 19 and Well 20). The microtiter plates were sealed andincubated for 1 hour at 37° C.

The microtiter plates were sealed and incubated for 45 min at 37° C.After the solution was removed from wells, the plates were washed fivetimes with2×SSC washing solution (0.3M sodium chloride, 0.03M tri-sodiumcitrate), 100 μl of alkaline phosphatase-conjugated streptavidin (GibcoBRL) solution, diluted to 1/1000 in TTBS enzyme diluting solution (0.2MTris-HCl(pH7.6), 0.5M sodium chloride, 0.5% Tween 20), was added to eachwell. After the solution was removed from wells, the plates were washedfive times with the washing solution described above, chromogenicsubstrate solution (4 mg/ml PNPP (p-nitrophenylphosphate), 1 mMmagnesium chloride, 10% diethanolamine (pH9.8)) was added and incubatedfor 30 min at 37° C. After incubation, color development was stopped byadding 25 μl of 0.5M EDTA to each well and the absorbance was measuredat 405 mm. The absorbance to each sequence is shown in Table 1. Theabsorbance of positive and negative signals was 1.0 and over, and under0.5, respectively. By using these results, HLA-A2 allele typing for eachsample (1-4) was performed according to the Typing Table shown inFIG. 1. The typing results are shown in the bottom column of Table 1 asfollows. TABLE 1 Results of HLA-A2 allele typing (the absorbance at 405nm) Well SS0 probe Sample 1 Sample 2 Sample 3 Sample 4 1 A240T 1.8941.907 2.049 1.849 2 A368A 1.675 1.744 0.116 1.210 3 A368G 0.265 0.2942.050 0.198 4 A368T 0.077 0.212 0.038 0.065 5 A362TT + 0.282 0.261 0.0520.202 A362TA 6 A98T 1.655 0.084 1.768 1.406 7 A98A 0.047 1.871 0.0381.589 8 A539T 1.952 1.971 1.974 1.127 9 A539A 0.267 0.280 0.380 0.232 10A402G 0.299 0.344 0.326 0.227 11 A527A 0.199 0.212 0.229 0.140 12 A270T0.194 0.265 0.263 0.229 13 A290T 0.118 0.104 0.105 0.112 14 A559G 0.0270.019 0.026 0.048 15 A485A 0.171 0.176 0.169 0.108 16 A355G 1.956 1.9711.877 1.344 17 A160A 0.024 0.024 0.030 0.030 18 A570CG 0.040 0.027 0.0500.064 19 A779A 0.020 0.021 0.034 0.041 20 A843A 0.025 0.049 0.038 0.045HLA-A2 A*0201 A*0206 A*0207 A*0201/ Allele type 0206

Example 2 HLA-B40 Allele Typing

Leukocytes (Samples 5-8) which were isolated from peripheral blood(about 10 ml) of normal subjects according to usual methods, were lysedin 500 μl of guanidine thiocyanate buffer(4M guanidine thiocyanate, 25mM sodium citrate(pH7.0), 0.5% sodium N-lauroylsarcosinate, 1%mercaptoethanol). The solution was extracted twice with phenol toeliminate proteins. After mixing with 3M sodium acetate buffer (pH 5.2,genome DNAs were obtained by adding twice volume of chilled ethanol. Byusing this DNAs, typing of the HLA-B40 alleles was performed as follows.

By using BASF-1 and 5′-biotinylated BASR-1 for a primer pair,amplification of the region containing the exon 2, the intron 2 and theexon 3 of the HLA-B40 alleles from DNAs described above was performed bythe PCR method. The reaction solution was composed of genomic DNAs (100ng), 1.4 units of thermostable DNA which was pretreated with TaqStart™Antibody for 5 min at room temperature, 33.5 mM Tris-HCl (pH 8.8),8.8 mM ammonium sulfate, 1.5 mM magnesium chloride, 0.005% Tween 20, 200μM dNTPs, and each 1.7 μM of a primer pair in a final volume of 70 μl.DNA amplification was performed by using GeneAmp PCR system 9600 (PerkinElmer) by initial denaturation at 95° C. for 2 min followed by 5 cyclesof denaturation for 25 s, annealing at 70° C. for 45 s, extension at 72°C. for 45 s followed by 36 cycles of denaturation for 25 s, annealing at65° C. for 50 s, extension at 72° C. for 45 s.

5′-amino-modified DNA probes, BL4, BL5, BL24, BL25, BL34, BL35, BL37,BL39, BL41, BL50, BL56, BL57, BL222A, BL409T and BL512T, wereimmobilized covalently on wells of carboxylate-modified polystyrenemicrotiter plates as follows. Twenty-five μl of the DNA probes describedabove which were dissolved with sterile distilled water, was added toeach of 15 wells which were used for a sample, in order shown in FIG. 2.Next, 75 μl of 0.2M EDC was added to each well and mixed. After theplates were sealed and incubated for 16 hours at room temperature, theywere washed four times with PBS buffer solution (7.5 mM di-potassiumhydrogenphosphate, 2.5 mM potassium dihydrogenphosphate, 0.15M sodiumchloride). Two hundreds μl of 0.4N NaOH were added to each well and theplates were incubated for 1 hour at 37° C. The plates were washed fourtimes with PBS buffer solution.

One hundred μl of GMC buffer (0.25M di-sodium hydrogenphosphate, 7% SDS,1% BSA, 0.5M EDTA, 0.03M phosphoric acid, 20% formamide) was added toeach well of the microtiter plates and the plates were incubated for 5min at 37° C. After incubation, the buffer was removed from each well.During incubation, 60 μl of the amplified products described above, weredenatured with an equivalent volume of 0.4 NaOH for 5 min at roomtemperature. After denaturation, 1500 μl of hybridization buffer wasadded to the denatured product, mixed and 100 μl of them was added toeach well. The microtiter plates were sealed and incubated for 1 hour at37° C.

After the solution was removed from wells, the plates were washed fivetimes with 2×SSC washing solution (0.3M sodium chloride, 0.03Mtri-sodium citrate), 100 μl of peroxidase-conjugated streptavidin(Vector Laboratories) solution, diluted to 1/2000 in TTBS enzymediluting solution (0.2M Tris-HCl(pH7.6), 0.5M sodium chloride, 0.5%Tween 20), was added to each well. The microtiter plates were sealed andincubated for 15 min at 37° C. After the solution was removed fromwells, the plates were washed five times with the washing solutiondescribed above, chromogenic substrate solution(3,3′,5,5′-tetramethylbenzidine (TMB) solution:Kirkegaard & PerryLaboratories) was added and incubated for 30 min at 37° C. Afterincubation, color development was stopped by adding 100 μl of 1% SDS toeach well and the absorbance was measured at 650 mm. The absorbance toeach sequence is shown in Table 2. The absorbance for positive andnegative signals was 1.0 and over, and under 0.5, respectively. By usingthese results, HLA-B40 allele typing for each sample (5-8) was performedaccording to the Typing Table shown in FIG. 2. The typing results areshown in the bottom column of Table 2 as follows. TABLE 2 Results ofHLA-B40 allele typing (the absorbance at 650 nm) Well SS0 probe Sample 5Sample 6 Sample 7 Sample 8 1 BL222A 1.846 1.671 1.742 1.849 2 BL34 2.1262.148 2.182 2.239 3 BL35 0.088 0.082 0.083 0.093 4 BL4 1.966 1.870 1.8001.976 5 BL5 0.154 0.161 0.142 0.205 6 BL24 1.711 1.744 1.671 2.018 7BL25 0.050 0.051 0.056 0.067 8 BL512T 2.356 0.209 0.238 0.058 9 BL370.130 2.533 2.517 0.014 10 BL39 0.069 0.099 0.111 0.027 11 BL41 0.0420.064 0.070 2.315 12 BL50 0.101 0.014 0.039 0.044 13 BL56 2.487 2.4640.373 2.342 14 BL57 0.193 0.156 2.124 0.093 15 BL409T 0.038 0.050 0.2870.031 HLA-B40 B*4001 B*4002 B*4003 B*4006 Allele type

Example 3 HLA-A Antigen and Allele Typing

Leukocytes (Samples 9-12) which were isolated from peripheral blood(about 10 ml) of normal subjects according to usual methods, were lysedin 500 μl of guanidine thiocyanate buffer (4M guanidine thiocyanate, 25mM sodium citrate(pH7.0), 0.5% sodium N-lauroylsarcosinate, 1%mercaptoethanol). The solution was extracted twice with phenol toeliminate proteins. After mixing with 3 M sodium acetate buffer (pH5.2),genome DNAs were obtained by adding twice volume of chilled ethanol. Byusing this DNAs, typing of the HLA-A antigens and alleles was performedas follows.

By using CGA011, CGA012 and 5′-biotinylated AIn3-66C for a primer pair,amplification of the region containing the exon 2, the intron 2 and theexon 3 of the HLA-A alleles from DNAs described above was performed bythe PCR method. The reaction solution was composed of genomic DNAs (100ng), 1.4 units of thermostable DNA polymerase which was pretreated withTaq Start™Antibody for 5 min at room temperature, 33.5 mM Tris-HCl (pH8.8), 8.8 mM ammonium sulfate, 1.5 mM magnesium chloride, 0.005% Tween20, 200 μM dNTPs, and each 1.7 μM of a primer pair (the ratio of CGA011to CGA012 is 4 to 1) in a final volume of 100 μl. DNA amplification wasperformed by using GeneAmp PCR system 9600 (Perkin Elmer) by initialdenaturation at 95° C. for 2 min followed by 5 cycles of denaturationfor 25 s, annealing at 70° C. for 45 s, extension at 72° C. for 45 sfollowed by 36 cycles of denaturation for 25 s, annealing at 65° C. for50 s, extension at 72° C. for 45 s.

5′-amino-modified DNA probes, A34, A239A, A238A, A257TC, A259AC, A282C,A282CT, A290TR, A299T, A302GR, A355G, A414A, A448C, A468T, A489A, A502C,A526T, A538CG, A538TG, A539A, A539T, A555T, A570CG and A570GT, wereimmobilized covalently on wells of carboxylate-modified polystyrenemicrotiter plates as follows. Twenty-five μl of the DNA probes describedabove which were dissolved with sterile distilled water, was added toeach of 23 wells which were used for a sample, in order shown in FIG. 3.Next, 75 μl of 0.2M EDC solution was added to each well and mixed. Afterthe plates were sealed and incubated for 16 hours at room temperature,they were washed four times with PBS buffer solution (7.5 mMdi-potassium hydrogenphosphate, 2.5 mM potassium dihydrogenphosphate,0.15M sodium chloride). Two hundreds μl of 0.4N NaOH were added to eachwell and the plates were incubated for 1 hour at 37° C. The plates werewashed four times with PBS buffer solution.

One hundred μl of GMC buffer (0.25M di-sodium hydrogenphosphate, 7% SDS,1% BSA, 0.5M EDTA, 0.03M phosphoric acid, 20% formamide) was added toeach well of the microtiter plates and the plates were incubated at 37°C. for 5 min. After incubation, the buffer of each well was removed fromeach well. During incubation, 96 μl of the amplified products describedabove, were denatured with an equivalent volume of 0.4 NaOH for 5 min atroom temperature. After denaturation, 2400 μl of hybridization bufferwas added to the denatured products, mixed and 100 μl of them was addedto each well. The microtiter plates were sealed and incubated for 1 hourat 37° C.

After the solution was removed from wells, the plates were washed fivetimes with 2×SSC washing solution (0.3M sodium chloride, 0.03Mtri-sodium citrate), 100 μl of peroxidase-conjugated streptavidin(Boehringer Mannheim) solution, diluted to 1/2000 in TTBS enzymediluting solution (0.2M Tris-HCl(pH7.6), 0.5M sodium chloride, 0.5%Tween 20), was added to each well. The microtiter plates were sealed andincubated for 15 min at 37° C. After the solution was removed fromwells, the plates were washed five times with the washing solutiondescribed above, chromogenic substrate solution (TMB solution:Kirkegaard & Perry Laboratories) was added and incubated for 30 min at37° C. After incubation, color development was stopped by adding 100 μlof 1% SDS to each well and the absorbance was measured at 650 mm. Theabsorbance for positive and negative signals was 1.0 and over, and under0.5, respectively. By using these results, HLA-A antigen and alleletyping for each sample (9- 12) was performed according to the TypingTable shown in FIG. 3. The typing results are shown in the bottom columnof Table 3 as follows. TABLE 3 Results of HLA-A antigen and alleletyping (the absorbance at 650 nm) Sample Well SS0 probe Sample 9 10Sample 11 Sample 12 1 A468T 2.963 3.046 2.603 2.719 2 A570CG 0.087 2.9510.081 2.847 3 A570GT 2.815 0.065 2.690 2.763 4 A282C + 1.950 2.825 2.5382.552 A282CT 5 A299T 0.111 0.119 0.279 0.162 6 A290TR 0.012 0.135 2.2450.095 7 A355G 2.382 0.033 0.037 0.128 8 A259AC 0.048 0.063 0.095 2.127 9A257TC 0.034 0.021 0.054 0.060 10 A238A −0.016 0.011 1.907 0.041 11A239A 0.037 0.052 0.061 0.187 12 A538CG 0.012 0.025 0.017 0.065 13 A555T0.068 0.038 0.066 0.090 14 A539T 2.480 0.048 1.618 0.093 15 A539A 0.1112.513 0.205 2.402 16 A526T 0.023 0.046 0.105 0.065 17 A538TG 0.109 0.1180.092 2.125 18 A302GR −0.020 0.169 0.030 0.237 19 A34 2.186 0.121 1.4412.271 20 A414A 0.031 0.127 0.079 0.095 21 A448C 0.232 0.091 0.073 2.41222 A489A 2.896 0.100 0.051 0.276 23 A502C 0.017 0.135 1.401 2.517 HLA-AA2/— A24/— A*31012/— A24/26 antigen and Allele type

Example 4 HLA-B Antigen and Allele Typing

Leukocytes (Samples 13-16) which were isolated from peripheral blood(about 10 ml) of normal subjects according to usual methods, were lysedin 500 l of guanidine thiocyanate buffer (4M guanidine thiocyanate, 25mM sodium citrate(pH7.0), 0.5% sodium N-lauroylsarcosinate, 1%mercaptoethanol). The solution was extracted twice with phenol toeliminate proteins. After mixing with 3 M sodium acetate buffer (pH5.2),genome DNAs were obtained by adding twice volume of chilled ethanol. Byusing the DNAs, typing of the HLA-B antigen and allele was performed asfollows.

By using 5BIN1-TA, 5BIN1-CG and 5′-biotinylated 3BIN3-37 for a primerpair, amplification of the region containing the exon 2, the intron 2and the exon 3 of the HLA-B alleles from DNAs described above wasperformed by the PCR method. The reaction solution was composed ofgenomic DNAs (100 ng), 1.4 units of thermostable DNA polymerase whichwas pretreated with Taq Start™Antibody for 5 min at room temperature, 67mM Tris-HCl (pH 8.8), 16.6 mM ammonium sulfate, 1.5 mM magnesiumchloride, 0.01% Tween 20, 10% DMS0, 200 μM dNTPs, and each 1.7 μM of aprimer pair (the ratio of 5BIN1-TA to 5BIN-CG is 2 to 3) in a finalvolume of 100 μl. DNA amplification was performed by using GeneAmp PCRsystem 9600 (Perkin Elmer) by initial denaturation at 95° C. for 2 minfollowed by 5 cycles of denaturation for 25 s, annealing at 70° C. for45s, extension at 72° C. for45 s followed by 36 cycles of denaturation for25 s, annealing at 65° C. for 50 s, extension at 72° C. for 45 s.

5′-amino-modified DNA probes, BL1, BL3, BL4, BL9, BL10, BL11, BL34,BL36, BL37, BL38, BL39R, BL40, BL41, BL42, BL77, BL78, BL79, BL226G,BL263T, BL272A, BL527A, BL538CG, BL538G and BL570GT, were immobilizedcovalently on wells of carboxylate-modified polystyrene microtiterplates as follows. Twenty-five μl of the DNA probes described abovewhich were dissolved with sterile distilled water, was added to each of23 wells which were used for a sample, in order shown in FIGS. 4 and 5.Next, 75 μl of 0.2M EDC was added to each well and mixed. After theplates were sealed and incubated for 16 hours, they were washed fourtimes with PBS buffer solution (7.5 mM di-potassium hydrogenphosphate,2.5 mM potassium dihydrogenphosphate, 0.15M sodium chloride). Twohundreds μl of 0.4N NaOH were added to each well and the plates wereincubated for 1 hour at 37° C. The plates were washed four times withPBS buffer solution.

One hundreds μl of GMC buffer (0.25M di-sodium hydrogenphosphate, 7%SDS, 1% BSA, 0.5M EDTA, 0.03M phosphoric acid, 20% formamide) was addedto each well of the microtiter plates and the plates were incubated for5 min at 37° C. After incubation, the buffer was removed from each well.During incubation, 96 μl of the amplified products described above, weredenatured with an equivalent volume of 0.4 NaOH for 5 min at roomtemperature. After denaturation, 2400 μl of hybridization buffer wasadded to the denatured products, mixed and 100 μl of them was added toeach well. The microtiter plates were sealed and incubated for 1 hour at37° C.

After the solution was removed from wells, the plates were washed fivetimes with 2×SSC washing solution (0.3M sodium chloride, 0.03Mtri-sodium citrate), 100 μl of peroxidase-conjugated streptavidin(Boehringer Mannheim) solution, diluted to 1/2000 in TTBS enzymediluting solution (0.2M Tris-HCl(pH7.6), 0.5M sodium chloride, 0.5%Tween 20), was added to each well. The microtiter plates were sealed andincubated for 15 min at 37° C. After the solution was removed fromwells, the plates were washed five times with the washing solutiondescribed above, chromogenic substrate solution (TMB solution:Kirkegaard & Perry Laboratories) was added and incubated for 30 min at37° C. After incubation, color development was stopped by adding 100 μlof 1% SDS to each well and the absorbance was measured at 650 mm. Theabsorbance for positive and negative signals was 1.0 and over, and under0.5, respectively. By using these results, HLA-B antigen and alleletyping for each sample (13 16) was performed according to the TypingTables shown in FIGS. 4 and 5. The typing results are shown in thebottom column of Table 4 as follows. TABLE 4 Results of HLA-B antigenand allele typing (the absorbance at 650 nm) Sample Sample Sample WellSS0 probe 13 14 15 Sample 16 1 BL36 0.064 0.131 0.101 0.087 2 BL37 2.1550.055 0.021 0.009 3 BL38 0.447 0.150 0.110 0.071 4 BL39R 0.147 1.4760.143 0.103 5 BL40 0.026 0.040 0.290 0.211 6 BL41 0.064 0.062 2.6502.213 7 BL42 0.268 0.235 0.237 0.120 8 BL77 2.564 0.038 0.075 0.128 9BL78 0.104 2.559 2.549 2.627 10 BL79 0.115 0.232 0.199 2.316 11 BL10.080 1.065 0.176 0.241 12 BL9 1.787 0.124 0.058 1.142 13 BL3 0.1730.163 0.141 0.144 14 BL4 0.055 1.720 0.142 0.215 15 BL10 2.256 0.0510.066 1.847 16 BL11 0.178 0.064 0.264 0.054 17 BL272A 0.038 0.105 0.0440.071 18 BL226G 0.034 0.163 0.137 0.102 19 BL263TA 0.005 0.173 0.0480.012 20 BL34 1.992 0.168 0.186 2.446 21 BL527A 2.674 0.383 2.369 1.94822 BL538CG + 2.619 0.311 0.354 0.356 BL538G 23 BL570GT 2.538 0.421 2.6452.821 HLA-B B7/— B*4403/— B51/— B51/55 antigen and allele type

Example 5 HLA-C Allele Typing

Leukocytes (Samples 17-20) which were isolated from peripheral blood(about 10 ml) of normal subjects according to usual methods, were lysedin 500 μl of guanidine thiocyanate buffer (4M guanidine thiocyanate, 25mM sodium citrate(pH7.0), 0.5% sodium N-lauroylsarcosinate, 1%mercaptoethanol). The solution was extracted twice with phenol toeliminate proteins. After mixing with 3M sodium acetate buffer (pH5.2),genome DNAs were obtained by adding twice volume of chilled ethanol. Byusing the DNAs, typing of the HLA-C alleles was performed as follows.

By using 5BCIn37-24C, 5BCIn-37-24g and 5′-biotinylated 5BCIn37-34g2 fora primer pair, amplification of the region containing the exon 2, theintron 2 and the exon 3 of the HLA-C alleles from DNAs described abovewas performed by the PCR method. The reaction solution was composed ofgenomic DNAs (100 ng), 1.4 units of thermostable DNA polymerase whichwas pretreated with Taq Start™Antibody for 5 min at room temperature,33.5 mM Tris-HCl (pH 8.8), 8.8 mM ammonium sulfate, 1.5 mM magnesiumchloride, 0.005% Tween 20, 200 μM dNTPs, and each 1.7 μM of a primerpair in a final volume of 100 μl. DNA amplification was performed byusing GeneAmp PCR system 9600 (Perkin Elmer) by initial denaturation at95° C. for 2 min followed by 5 cycles of denaturation for 25 s,annealing at 70° C. for 45 s, extension at 72° C. for 45 s followed by36 cycles of denaturation for 25 s, annealing at 65° C. for 50 s,extension at 72° C. for 45 s.

5′-amino-modified DNA probes, 201g1, C206gR, A-12, RA-2, A-3, RA-41,A-54, B-1, RB-28, C-12, C-24, C-33, C-43, 134-g, 134-A2, 353TCA1, R341A,343 A, R343g3, 353TCC, 361T1, 361T368g, 361T368T1, 369C, 387gl, 526AC2and 538gAC, were immobilized covalently on wells of carboxylate-modifiedpolystyrene microtiter plates as follows. Twenty-five μl of the DNAprobes described above which were dissolved with sterile distilledwater, was added to each of 23 wells which were used for a sample, inorder shown in FIG. 6. Next, 75 μl of 0.2M EDC solution was added toeach well, mixed and sealed. After the plates were sealed and incubatedfor 16 hours, they were washed four times with PBS buffer solution (7.5mM di-potassium hydrogenphosphate, 2.5 mM potassium dihydrogenphosphate,0. 15M sodium chloride). Two hundreds μl of 0.4N NaOH were added to eachwell and the plates were incubated for 1 hour at 37° C. The plates werewashed four times with PBS buffer solution.

One hundred μl of GMC buffer (0.25M di-sodium hydrogenphosphate, 7% SDS,1% BSA, 0.5M EDTA, 0.03M phosphoric acid, 20% formamide) was added toeach well of the microtiter plates and the plates were incubated for 5min at 37° C. After incubation, the buffer was removed from each well.During incubation, 96 μl of the amplified products described above, weredenatured with an equivalent volume of 0.4 NaOH for 5 min at roomtemperature. After denaturation, 2400 μl of hybridization buffersolution was added to the denatured products, mixed and 100 μl of themwas added to each well. The microtiter plates were sealed and incubatedfor 1 hour at 37° C.

After the solution was removed from wells, the plates were washed fivetimes with 2×SSC washing solution (0.3M sodium chloride, 0.03Mtri-sodium citrate). One hundred μl of peroxidase-conjugatedstreptavidin (Boehringer Mannheim) solution, diluted to 1/2000 in TTBSenzyme diluting solution (0.2M Tris-HCl(pH7.6), 0.5M sodium chloride,0.5% Tween 20), was added to each well. The microtiter plates weresealed and incubated for 15 min at 37° C. After the solution was removedfrom wells, the plates were washed five times with the washing solutiondescribed above, chromogenic substrate solution (TMB solution:Kirkegaard & Perry Laboratories) was added and incubated for 30 min at37° C. After incubation, color development was stopped by adding 100 μlof 1% SDS to each well and the absorbance was measured at 650 mm. Theabsorbance for positive and negative signals was 1.0 and over, and under0.5, respectively. By using these results, HLA-C allele typing for eachsample (17-20) was performed according to the Typing Table shown in FIG.6. The typing results are shown in the bottom column of Table 5 asfollows. TABLE 5 Results of HLA-C allele typing (the absorbance at 650nm) Well SS0 probe Sample 17 Sample 18 Sample 19 Sample 20 1 C206gR2.080 2.069 2.003 1.871 2 A-12 2.165 −0.024 −0.029 1.805 3 RA-2 0.0201.992 0.120 1.979 4 A-3 0.069 0.038 0.052 0.081 5 RA-41 0.008 0.0330.121 0.102 6 A-54 −0.012 0.194 2.080 0.059 7 B-1 0.202 0.124 0.1450.233 8 RB-28 2.403 1.640 1.716 1.998 9 C-12 1.855 0.045 0.019 1.739 10C-24 0.138 0.064 2.002 0.287 11 C-33 0.086 2.563 0.077 2.181 12 C-430.113 0.182 0.137 0.174 13 134-g 1.594 0.089 1.763 1.384 14 134-A2 0.0492.096 0.291 1.380 15 343A 0.021 2.672 0.047 1.480 16 R343g3 + 2.5620.292 2.717 1.928 R341A 17 353TCA1 0.001 2.551 0.157 1.740 18 353TCC0.021 −0.002 0.092 0.006 19 201g1 1.209 1.679 0.176 1.225 20 369C 0.0550.183 2.640 0.163 21 361T1 + 2.345 0.040 0.048 1.885 361T368g +361T368T1 22 387g1 0.028 0.054 0.015 0.019 23 526AC2 + 0.090 0.074 0.1240.092 538gAC HLA-C allele type C*0102/— C*0303/— C*1202/— C*0102/ 0303Industrial Applicability

By this invention, a single HLA class I antigen or allele is determinedby combining PCR amplification using a primer pair which can amplify allthe HLA-A alleles, all the HLA-B alleles or all the HLA-C alleles orwhich is specific to the common sequence to alleles of the specificgroup consisting of the specific HLA-A alleles or the specific HLA-Balleles, with reverse hybridization analysis using DNA probes to enableto specifically hybridize with the sequence of al least a specific HLA-Aallele, at least a specific HLA-B allele or at least a specific HLA-Callele, which are covalently immobilized on wells of microtiter plates.Therefore, it can solve problems from the viewpoint of manipulation ofHLA class I loci antigen typing by the classical serological method, andcan classify at the allele level (allele typing) the class I antigens orsubtypes to be unable to distinguish and classify by the classicalmethod. Furthermore, at the same time, it can solve problems from theviewpoint of manipulation and resolution of HLA class I allele typing.Namely, this invention enables us to easily mechanize and automatedetection and determination of the HLA class I alleles. This inventionoffers a method, a reagent and a kit for typing of the HLA class Ialleles, which are useful for judgement of compatibility between a donorand a recipient in organ transplantation and for association analysisbetween the HLA class I genes and various kinds of diseases in theclinical and medical field.

1. A method for detecting a specific base sequence, whereinhybridization is performed in a hybridization buffer containing 10% to25% formamide, at 32° C. to 42° C., using a probe of 14 to 24 or more ofbases.
 2. The method claimed in claim 1, wherein the hybridizationbuffer contains 0.25M di-sodium hydrogenphosphate, 7% sodium dodecylsulfate, 1% bovine serum albumin, 0.03M phosphoric acid, 0.5Methylenediaminetetraacetic acid and 10% to 25% formamide.
 3. The methodclaimed in claim 1 or 2, wherein the temperature for washing after thehybridization is room temperature.
 4. The method claimed in claim 1,wherein the probes are hybridized with amplified products by the PCRmethod.
 5. The method claimed in claim 4, wherein at least one of theprimer pair is labeled.
 6. The method claimed in claim 1, whereinnucleic acids are hybridized with the probes immobilized on a support.7. The method claimed in claim 4, which comprises hybridizing theamplified products obtained by the PCR method with the immobilized DNAprobes, adding an enzyme-conjugate which specifically bonds to a labelof the amplified products thereto at the same time or after thehybridization, and adding a chromogenic substrate, a luminescentsubstrate or a fluorescent substrate to the mixture, to detect assignals whether or not the amplified products are hybridized with theimmobilized DNA probes.
 8. The method claimed in claim 7, wherein thelabel is a biotin and the enzyme-conjugate is an enzyme-conjugatedstreptavidin.